The present description refers to photocleavable compounds which can be used as a photocleavable linker in order to link two biomolecules, such as oligonucleotides and peptides. The present description further refers to a method for the synthesis of said photocleavable compounds.
Photocleavable compounds play an important role as protecting groups in blocking functional groups present in nucleosides, nucleotides, sugars and amino acids, which are used for the synthesis of biomolecules, e.g. nucleic acids and their derivatives, proteins, peptides and carbohydrates. Such compounds have the advantage that deprotection of the protected functional group can be performed simply via light exposure. Therefore, photocleavable compounds provide the basis for the photolithography based spatially resolved synthesis of oligonucleotides or peptides on solid supports, such as microarrays. The use of photolabile compounds for the synthesis of microarrays is well known (Pease, et al. Proc. Natl. Acad. Sci. USA 91 (1994) 5022-5026), Hasan, et al. Tetrahedron 53 (1997) 4247-4264).
Photocleavable compounds can also be used as photocleavable linker molecules to link different elements, such as two biomolecules, a biomolecule and a solid phase, a biomolecule and streptavidin. For example, two different oligonucleotides may be connected via the photocleavable compound in order to deliver a single stranded molecule to a biochemical or biological assay, which upon irradiation with light releases the two individual oligonucleotides which then may form a double stranded DNA and exhibit biological function. Another possible application is that an oligonucleotide may be linked to a biotin molecule via a photocleavable compound such that the oligonucleotide may be captured by affinity purification, e.g. by streptavidin coated magnetic beads from an assay and secondly may be released from the affinity resin by irradiation with light.
Yet another possible application is that the aforementioned oligonucleotide-photocleavable-compound-biotin structure may be useful in pull-down purifications or assays, when the oligonucleotide is a protein binding aptamer.
The elements, which are photocleavably connected by the above mentioned compounds can be separated from each other simply due to light exposure. Such components are well known (Olejnik, et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7590-7594; Olejnik, et al. Nucleic Acids Res. 26 (1998) 3572-3576; Olejnik, et al. Nucleic Acids Res. 27 (1999) 4626-4631).
The major drawback of photocleavable compounds described in the art is that light in the near UV-range (365 nm or shorter) has to be used for cleaving the covalent bond(s) in order to cleave the photocleavable compound. Light sources, which are suitable to generate such wavelength, are e.g. mercury arc lamps, excimer lasers, UV-LEDs and frequency multiplied solid-state lasers. Such light sources are characterized by high purchase costs, provide limited luminous power and have a short life-time leading to high overall costs of operation. Since some of the above mentioned light sources contain hazardous substances, e.g. mercury, appropriate actions to secure occupational safety and proper disposal are necessary further increasing the costs.
Some of the above mentioned light sources produce a broad spectrum of wavelengths, e.g. mercury arc lamps emit light from the UV- to the IR-range, both of which have disadvantageous effects if used in the context of biomolecules. UV-light for example can be absorbed by the synthesized DNA leading to random breaks within the strand by phosphate backbone radical cleavage, guanine base oxidation and subsequent strand break or photodimerization, especially of thymine bases. Furthermore, UV-light can also lead to the destruction of certain amino acids, such as tryptophan by radical oxidation or cysteine and methionine by sulfur oxidation. As a result, experiments involving biomolecules are influenced by the use of such UV-light for the cleavage of the photocleavable compounds. In contrast, IR-light leads to warming of the experimental setup which also influences the results of the experiments.
The object of the present description is therefore the provision of photocleavable compounds, which do not show the above mentioned drawbacks.